Longitudinal analysis of antibody responses to Plasmodium vivax sporozoite antigens following natural infection

Background P. vivax malaria is a major global health burden hindering social and economic development throughout many tropical and sub-tropical countries. Pre-erythrocytic (PE) vaccines emerge as an attractive approach for the control and elimination of malaria infection. Therefore, evaluating the magnitude, longevity and prevalence of naturally acquired IgG antibody responses against PE candidate antigens is useful for vaccine design. Methodology/Principal findings The antigenicity of five recombinant PE antigens (PvCSP-VK210, PvSSP3, PvM2-MAEBL, PvCelTOS and PvSPECT1) was evaluated in plasma samples from individuals residing in low transmission areas in Thailand (Ranong and Chumphon Provinces). The samples were collected at the time of acute vivax malaria and 90, 270 and 360 days later. The prevalence, magnitude and longevity of total IgG and IgG subclasses were determined for each antigen using the longitudinal data. Our results showed that seropositivity of all tested PE antigens was detected during infection in at least some subjects; anti-PvCSP-VK210 and anti-PvCelTOS antibodies were the most frequent. Titers of these antibodies declined during the year of follow up, but notably seropositivity persisted. Among seropositive subjects at post-infection, high number of subjects possessed antibodies against PvCSP-VK210. Anti-PvSSP3 antibody responses had the longest half-life. IgG subclass profiling showed that the predominant subclasses were IgG1 and IgG3 (cytophilic antibodies), tending to remain detectable for at least 360 days after infection. Conclusions/Significance The present study demonstrated the magnitude and longevity of serological responses to multiple PE antigens of P. vivax after natural infection. This knowledge could contribute to the design of an effective P. vivax vaccine.


Introduction
Plasmodium vivax is one of the main agents responsible for malaria and high morbidity and mortality [1].P. vivax has distinct biological characteristics, including a dormant hypnozoite stage in the liver that can cause relapse and the early production of gametocytes that promote further transmission [2].Unfortunately, genetic study revealed that the prevalence of drug-resistant strains of P. vivax is increasing at an alarming rate, thereby making the control, elimination and eradication of malaria difficult [3].This necessitates the development of a long-lasting and fully effective vaccine against P. vivax to accelerate its elimination [4,5].
Nowadays, pre-erythrocytic (PE) or anti-infective vaccines stand out as the most attractive approach for malaria prevention, by targeting parasites at the early phase of infection and reducing the severity of clinical symptoms.Experimental evidence in P. falciparum infections demonstrates that repeated exposure to high doses of chemically-, genetically-or radiationattenuated sporozoites can induce protection [6][7][8].However, a phase III clinical trial of the PE vaccine RTS,S/ASO1 showed limited efficacy and relatively short-lived protection [9][10][11].To improve this efficacy, a second generation of P. falciparum CSP-based subunit vaccine was designed to promote the development of long-lived, affinity-matured plasma cells that maintain protective titers of anti-sporozoite antibody.In contrast to P. falciparum, very few P. vivax PE vaccines have progressed to clinical trials [12][13][14].Therefore, a deeper understanding of the humoral responses against P. vivax PE antigens may help in the assessment of their potential as candidate vaccine antigens.PE antigens have been selected as potential vaccine candidates based on their involvement in sporozoite motility and hepatocyte invasion, and being targets of neutralizing antibodies.These include Circumsporozoite protein (CSP) [15], Sporozoite surface protein 3 (SSP3) [16], Sporozoite protein essential for cell traversal 1 (SPECT1) [17,18] and Membrane-associated erythrocyte binding-like protein (MAEBL) [19].CSP and SSP3 are surface-exposed sporozoite antigens and it is postulated that they become easily recognizable by immune antibodies, thereby blocking sporozoite invasion into erythrocytes.CSP is the dominant sporozoite surface protein and the long-time leading PE vaccine candidate [20].Previous studies revealed that irradiated sporozoites induce CSP-specific immune responses which are associated with sterile immunity [21][22][23].Even though P. falciparum circumsporozoite protein (PfCSP) is incorporated in the RTS,S vaccine and shows high efficacy in some settings [24], P. vivax CSPbased vaccines did not show satisfactory levels of protective efficacy in initial clinical trials [25][26][27].SSP3 was found to be essential for sporozoite motility and infectivity [16,28].Although there is no report associating naturally acquired immunity to SSP3 with protection, genetic analysis has shown that PbSSP3 -mutant sporozoites have decreased exoerythrocytic maturation [28].The sporozoite micronemal (MAEBL) and traversal proteins (Cell-traversal protein for ookinetes and sporozoites; CelTOS and Sporozoite protein essential for cell traversal 1; SPECT1) are also promising PE vaccine candidates.MAEBL is a sporozoite attachment protein vital for mosquito salivary gland infection and hepatocyte invasion [19,[29][30][31][32]. Anti-sera targeting the ectodomain M2 of MAEBL demonstrated inhibition of PE development and lethal P. yoelii infection [29,30].CelTOS play a crucial role in cell-traversal ability [33].A study of P. berghei celtos genetic disruption found that the absence of this gene reduced sporozoite infectivity in the liver and significantly inhibited cell traversal by ookinetes in mosquito midgut wall and sporozoite traversal of different kinds of cells [34].SPECT1 is a PE immune target due to the key role it plays in the malaria parasite's crossing the dermis and liver sinusoidal wall prior to invasion of hepatocytes [35][36][37].Up to now, none of the PE vaccines tested achieved satisfactory rates of protection, and the induced responses tended to be short-lived or highly strain specific.As such, it is of great importance to further assess the magnitude and durability of antibodies against these PE antigens in P. vivax-infected subjects to determine whether they have sufficient immunogenicity to enhance and prolong the protective effects of PE vaccines.
In the present study, we sought to assess the prevalence, magnitude and durability of antibody responses against five different PE antigens: PvCSP-VK210, PvSSP3, PvSPECT1, PvM2-MAEBL and PvCelTOS.The half-life of each PE antigen-specific total IgG was estimated using exponential curve fitting models.Moreover, the magnitude and longevity of IgG subclass antibodies against antigens that showed the highest antigenicity (PvCSP-VK210 and PvSSP3) were determined.Details of antibody responses to PE antigens are beneficial to evaluate these antigens as candidate vaccines against vivax malaria.

Ethics statement
This study was approved by the Committee on Human Rights Related to Human Experimentation, Mahidol University Central Institutional Review Board (MU-IRB 2012/079.2408and MU-CIRB 2021/281.2505).Written informed consent was obtained from each participant before blood collection.The criteria for recruitment of vivax malaria subjects were: (1) systolic blood pressure higher than 90 mmHg, (2) body temperature lower than 40˚C, (3) hematocrit higher than 25%, and (4) age 18 years or above.Individuals who did not meet these criteria were excluded.

Study site and participants
Heparinized blood samples were taken from subjects in malaria low-transmission areas in the southern part of Thailand (Chumphon and Ranong Provinces).Acute blood samples were collected at the Ministry of Public Health malaria clinics (Vector Borne Disease Units 11.4.2 and 11.5.3) and convalescent samples (day 90, 270 and 360) were collected at participants' residences from August 2017 to December 2022.To assess naturally-acquired IgG responses to P. vivax PE antigens, a cross-sectional study was conducted that included P. vivax-infected subjects during at acute phase or day 0 (n = 104) and P. vivax subjects who recovered from infection for 90 days (n = 41), 270 days (n = 30) and 360 days (n = 27).The acute subjects who showed seropositive responses to PvCSP-VK210 (n = 35) or PvSSP3 (n = 28) were selected for assessment of IgG subclass responses.Of the 104 acute-phase subjects, 20 individuals were selected in cohort study for monitoring anti-PE antibody titers at four studied time points (days 0, 90, 270 and 360).Of these 20 subjects in 1-year cohort study, 10 and 8 subjects were determined to have detectable levels of anti-PvCSP-VK210 and -PvSSP3 IgG subclass antibodies at post-infection, respectively.The demographic information of recruited subjects is summarized in S1 Fig and S1 Table .All P. vivax infections in study subjects were documented microscopically by both thin and thick Giemsa-stained blood smears, and confirmed by nested PCR [38].The patients who had symptomatic and positive both P. vivax parasitemia and PCR were enrolled as acutely-infected patients (n = 104).The recovery P. vivax subjects at 90 (n = 41), 270 (n = 30) and 360 days (n = 27) after infection in cross-sectional study were tested for blood smear and PCR before blood collection.Histories of prior malaria infections in subjects were obtained from the records of malaria clinics.All participants were treated with a full course of Primaquine and Chloroquine.In cohort study, blood samples from P. vivax-infected subjects were collected and tested by nested PCR every three months to detect sub-patent malaria.Staff conducted weekly house-to-house visits to estimate the incidence of clinical malaria over the study period.Blood samples from malaria-naïve, healthy subjects (n = 52) who lived in a non-malaria endemic area (Bangkok, Thailand) were collected and used as healthy controls (HCs).

Detection of total IgG against PE antigens
Levels of total IgG antibodies specific to PE antigens (PvCSP-VK210, PvSSP3, PvM2-MAEBL, PvCelTOS and PvSPECT1) were determined by indirect ELISA as previously reported [40].Briefly, recombinant PE antigens at 2 μg/mL were coated on 96-well plates.Plasma (1:200 dilution) was added to wells followed by HRP-conjugated goat anti-human IgG (KPL, USA).Signal was developed with TMB substrate and read at 450 nm.Cut-off values were calculated using mean OD plus 2 SD of HCs (n = 52).Seropositivity was defined as a reactivity index (RI) greater than 1.0.For each plate, the study plasma and control samples (negative and positive controls) were incubated in the wells.Negative control wells were plasma of HCs (n = 2); positive control wells were plasma of P. vivax-infected subjects (n = 2) who showed the highest RI values.For kinetic studies, plasma from the same subject at different times were incubated on the same plate.All the samples were assessed in duplicate and mean values were used for analyses.Tetanus toxoid (Merck Millipore, Germany) was used as control protein for the detection of total IgG in P. vivax subjects and HCs and for the estimation of anti-PE antibody half-lives.

Analysis of IgG subclass responses to PvCSP-VK210 and PvSSP3
To identify the predominant IgG subclass responses to PvCSP-VK210 and PvSSP3, indirect ELISAs were used for detection of IgG1, IgG2, IgG3 and IgG4 antibodies specific to these two antigens.The 96-well plates were coated with 2 μg/ml recombinant antigen.Plasma samples were added at a dilution of 1:100 and incubated for 1 hour at RT.After washing, mouse antihuman antibodies at dilution 1/500 for IgG1 and IgG3, and 1/1000 for IgG2 and IgG4 (Invitrogen Corp, CA, USA) were added and incubated for 1 hour at RT.Then, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Biolegend, USA) at 1/500 for IgG1 and IgG3, and at 1/1,000 for IgG2 and IgG4, were used for detection.The OD of the reaction was measured at 450 nm after the addition of the tetramethylbenzidine (TMB) enzyme substrate (Sigma Aldrich, USA).The positive controls were plasma (n = 2) from the P. vivax-infected patient who had the highest OD value for total IgG.A baseline OD was established using samples from HCs (n = 18).The RIs of IgG subclasses specific to PvCSP-VK210 and PvSSP3 were obtained by dividing the mean sample OD by the cut-off value.The cut-off values (mean OD + 2 SD) were calculated from a set of negative controls obtained from HC samples.A sample was defined as positive for a specific antibody if the RI was is equal to or greater than 1.All samples were assessed in duplicate and mean values were used for analyses.

Data analysis
Statistical analyses were performed using GraphPad Prism version 8.4.3, (GraphPad Software, USA, https://www.graphpad.com).Statistical testing of serologic responses was performed using one-way ANOVA and Dunn's multiple comparison tests.Statistical significance was considered as p < 0.05.Data analysis was performed using R version 4.1.1.To perform linear mixed effects regression, lme4 package was used and antibody responses were normalized as the percentage of maximal antigen-specific total IgG responses across all time points and plotted against days after maximal reactivity.Adjusted R 2 (R 2 adj) represents goodness of fit.Halflife was calculated by the following formula: In(0.5)/estimate.For correlation analysis, corrplot package version 0.90 was used for plotting and data was clustered by ward.D2 hierarchical agglomerative clustering method [41].Heatmaps representing longitudinal serological responses and exponential curve fitting plots were made using ggplot2 package, version 3.3.5.

Magnitude and seroprevalence of antibody responses to P. vivax PE antigens
The magnitude and seroprevalence of antibodies against PE antigens were cross-sectionally assessed using plasma samples from P. vivax-infected patients at day 0 and after recovery on days 90, 270 and 360 (Fig 1).During acute phase, antibody responses (mean±SD) to full-  During the recovery phase, a significant decrease was observed in antibody response to fulllength CSP-VK210 at day 360 (1.17±0.45,p<0.05), while those to its N-and C-terminals showed no significant reduction (Fig 1B -1D).Interestingly, the levels of anti-SSP3 antibodies were stably maintained for up to 12 months post-infection (Fig 1E).Next, antibody responses to micronemal and traversal antigens were assessed (Fig 1F -1H).Antibody responses to PvM2-MAEBL (0.82±0.27) and PvSPECT1 (0.56±0.24) had decreased significantly (p<0.005 for both) by day 270 compared to those during acute malaria illness.Anti-PvCelTOS antibody titers were markedly reduced at day 90 (0.94±0.34, p<0.0001).

Correlations among antibody responses specific to PE antigens
To identify correlations between antibody responses to different PE antigens, and at different timepoints (days 0, 90, 270 and 360), the responses were pairwise compared (Fig 2 ).A strong correlation was defined as a Spearman's ρ greater than 0.6.At acute phase (day 0), PvSSP3 was positively and significantly correlated with PvM2-MAEBL (ρ = 0.

Longitudinal seropositivity rates of anti-PE antibodies
Seropositivity of anti-PE antibodies was determined after P. vivax infection in the cross-sectional survey.We then evaluated the longevity of seropositivity against PE antigens in twenty P. vivax-infected subjects whose plasma samples were available from each of the 4 timepoints (Fig 3A).Serological responses were considered long-lasting in subjects who remained seropositive for the whole 360 days.At acute phase (day 0), all subjects showed seropositive responses against full-length PvCSP-VK210, 85% for N-terminal PvCSP-VK210, and 90% for C-terminal PvCSP-VK210.After infection, seropositivity against PvCSP-VK210 decreased; 60% (12/20), 10% (2/20) and 15% (3/20) of subjects had persistent antibody responses to fulllength, N terminal and C-terminal PvCSP-VK210 antigens through day 360, respectively (Fig 3B).Fifteen (75%) subjects were PvSSP3 seropositive at day 0, whereas only 6 (30%) subjects remained antibody positive at day 360.For micronemal antigens, 80%, 75% and 45% of P. vivax-infected-subjects produced antibody responses at day 0 to PvM2-MAEBL, PvCelTOS and PvSPECT1, respectively (Fig 3B).Persisting positivity was the antibody levels in 30% and 10% of subjects to PvM2-MAEBL and PvCelTOS at day 360, respectively.A great reduction of antibody to PvSPECT1 was seen post-infection, and all subjects were seronegative at day 360 (Fig 3B).In addition, there were 5 acutely infected subjects showed seropositive response to 5 PE antigens and two subdomains of PvCSP-VK210.These subjects did not remain seropositive to all tested PE antigens; only anti-PvCSP-VK210 antibodies displayed seropositivity at 1-year post-infection (Fig 3B).

The anti-PE antibody half-lives
Since detectable anti-PE antibody was observed through day 360 in some P. vivax-infected subjects followed longitudinally, the half-lives of antibody responses were calculated.We employed a linear mixed-effect model using the reactivity index of each antigen as dependent variable, timepoint (days) as fixed effect, and subjects as random effect (Table 1).The analysis showed that SSP3 had the longest half-life (799 days, 95% CI = 686-961 days), while CSP-full length had the shortest (231 days, 95% CI = 191-291 days).The N-terminal and C-terminal PvCSP-VK210 antigens had half-lives of 443 days (95% CI = 372-521 days) and 323 days (95%

Discussion
Insights into the serological responses against P. vivax PE antigens could provide the benefit in the development of anti-Plasmodium vivax vaccines [15,45,46].Here, antibody responses against five PE candidates (PvCSP-VK210, PvSSP3, PvM2-MAEBL, PvCelTOS and PvSPECT1) and two subdomains of PvCSP-VK210 (N-terminal and C-terminal regions) were assessed in P. vivax patients during and after acute malaria infection.Our results showed the highest frequency of positive response during acute infection was to full-length PvCSP-VK210 (70%), compared to other PE antigens.We surveyed seroprevalence of anti-PvCSP-VK210 antibodies at recovery phase (Days 90, 270, 360) in comparison with acute phase of infection.At 360-day post-infection, anti-full-length PvCSP titers were significantly decreased, whereas antibody titers to N-and C-terminal did not greatly reduce.Of the 20 acute subjects in 1-year cohort study, 12 subjects without re-infection maintained positive IgG responses for at least one year.The calculated half-life of anti-PvCSP-VK210 antibodies was between 191 and 291 days.Similarly, high prevalence of PvCSP-VK210 seropositivity were also observed in regions of Brazilian Amazon and Western Thailand and high frequency of responders was consistent with the predominance of CSP-VK210 variants of P. vivax parasites in endemic areas [45][46][47].A study by Longley et.al. showed a stable anti-PvCSP IgG titer for over a year in absence of detectable blood stage P. vivax infections [46].Moreover, IgG responses to PvCSP-VK210 demonstrated that most of seropositive responses in symptomatic patients had previous P. vivax infection.Here, very low baseline of pre-existing anti-PvCSP antibodies in endemic villagers who lived in endemic areas with the absence of infection was detected and all samples showed seronegative responses with average RI (SD) of 0.63 (0.18) compared to 0.60 (0.20) of HCs.Altogether, our data support the development and persistence of IgG antibody responses to PvCSP following natural P. vivax infections.
Given the high immunogenicity of PvCSP, this antigen is potential to be included as a component in PE-based vaccine formulation.However, there are several gaps which must be addressed: (i) the prevalence of PvCSP allelic variants in endemic areas and its relationship with host immune responses as well as the contribution of pre-existing immune response to endemic strains in the variation of anti-PvCSP antibody responses; (ii) the inhibitory efficiency of anti-PvCSP antibody from high responders in inhibition of liver stage development.This might reflect the association between high antibody titers and malaria protection; (iii) given a higher frequency of antibody responses to full-length PvCSP compared to N-and C-termini, future studies should aim to identify target epitopes of naturally acquired anti-PvCSP inhibitory antibodies whether the predominant B epitopes are located at repeated region, as it was reported in P. falciparum CSP (PfCSP) [48]; (iv) since high antigenic diversity in C-terminal PfCSP displayed limited breadth of antibody response and evasion of cellular immunity [49], identification of T cell epitope on N-or C-subdomain of PvCSP and determination of the effect of these two subdomain polymorphisms on driving immune selection leading to strainspecific immunity are required; (v) the ability of PvCSP to induce development and persistence of long-lived memory B cell and plasma cell as well as its contribution to magnitude and durability of IgG responses.Our study in similar cohort of participants showed an expansion of atypical memory B cells during acute malaria and this MBC subset were persisted up to 3 months after parasite clearance [50], thus, it might account for the impairment of anti-PvCSP persistent responses.Together, a deeper understanding of immune responses to PvCSP could provide novel insights into vaccine design in the future.
The identification of additional sporozoite proteins that might be combined with PvCSP is of prime importance for a multi-component PE vaccine.Plasmodium yoelii sporozoite surface protein (PySSP3) is a newly characterized protein known to be essential for gliding motility [16].Immunization of mice with PySSP2 and PyCSP is completely protective against the challenge with P. yoelii [51,52].Here, anti-PvSSP3 seropositivity (43%) was detected during P. vivax infection and responses to this antigen were observed (37%) 360 days after infection.Anti-PvSSP3 antibody titers were stable in 30% (6/20) of subjects.Among tested PE antigens, PvSSP3 had the longest half-life (> 700 days).In addition, strong correlations were shown between antibody responses at day 0 to PvSSP3 and both PvCelTOS (Spearman ρ = 0.63) and PvM2-MAEBL (Spearman ρ = 0.70).At day 360, strong correlations between antibodies to PvSSP3 and both N-and C-terminal PvCSP (Spearman ρ = 0.60-0.63)were found.Together, our results indicate the immunogenicity of PvSSP3 to stimulate humoral immunity during infection.To address long-lasting anti-PvSSP3 antibody responses, further analysis on the stability of antibody responses and the generation of MBCs specific to this antigen in longitudinal studies are needed to support the half-life estimation.Possible explanations of correlation between anti-PvSSP3, anti-PvM2-MAEBL and anti-PvCelTOS antibodies that might require further investigation including: (i) the similarity of amino acid sequences that leads to stimulation of cross-reactive antibodies during infection; (ii) the timing of antigen release during parasite infection may be related.The PvSSP3-PvCSP correlation might indicate a co-localization of these antigens on the sporozoite surface with parallel induction of long-term antibody responses.Prior to inclusion of PvSSP3 in a PE vaccine, more studies of its molecular function and structural characteristics are required.The sporozoite's complexity and the expression of its various proteins contribute to its distinct biological functions.Here, sporozoite antigens which play roles in the traversal process (PvM2-MAEBL, PvSPECT1 and PvCelTOS) were assessed their antigenicity in vivax malaria patients.During acute infection, our results showed that the highest frequency of seropositivity was to PvCelTOS (70%), compared to PvM2-MAEBL (48%) and PvSPECT1 (30%).This was similar to findings in a previous study that showed naturally induced IgG responses to PvCel-TOS in P. vivax patients [46].Of these three antigens, anti-PvCelTOS antibody levels were higher than those specific for PvM2-MAEBL and PvSPECT1.In a year-long cohort study, only a few subjects maintained positive anti-PvCelTOS (10%) and anti-PvM2-MAEBL (30%) antibody titers, and none of the subjects had stable anti-PvSPECT1 responses.Antibody kinetics specific to PvCelTOS and PvM2-MAEBL demonstrated long half-lives (> 500 days).Together, our results indicated that while PvCelTOS has the ability to stimulate host immune responses in natural infection, the use of this antigen in PE vaccine design should consider the limited duration of its antibody response.
In the present study, assessment of IgG subclass responses to PvCSP-VK210 and PvSSP3 revealed that the major subclasses were cytophilic antibodies IgG1 and IgG3, with limited IgG2 and IgG4 responses.These cytophilic antibodies made up the preponderant antibody response, similar to previous studies using PvCSP and PvTRAP [45] and in P. falciparum asexual blood stage antigens [44].Upon RTS/S vaccination, cytophilic IgG3 levels are associated with protection, followed by IgG1 levels [42].A cohort study in Papua New Guinea (PNG) children revealed an acquisition of cytophilic IgG responses to P. falciparum reticulocyte binding-like homologue protein (PfRH5).The high IgG3 responders to this antigen had a stronger association with reduced risk of malaria during 1-year monitoring period [44].From our result, the prevailing immune response involving cytophilic subclasses directed at sporozoite surface proteins, specifically PvCSP and PvSSP3, might serve a protective role against malaria through complement-mediated lysis [53] or cell-mediated mechanisms, such as opsonic phagocytosis and antibody-dependent cellular inhibition [54].Accordingly, inclusion of PvCSP and PvSSP3 in PE vaccine design may induce the magnitude and persistence of IgG1 and IgG3 responses which effectively kill the sporozoites.
Here, we demonstrated the ability of P. vivax subjects to produce anti-PE antibodies against all tested PE antigens.Of 104 P. vivax acute subjects, 67 samples (64.4%) responded to at least three of PE antigens.Nine acutely infected patients (8.7%) produced antibodies against all tested PE antigens.In 1-year cohort study, 25% (5/20) of subjects showed seropositivity to five PE antigens and two PvCSP-VK210 subdomains during acute infection, whereas these subjects maintained seropositive responses to only PvCSP-VK210 for one-year period.Together, our study demonstrated an ability of symptomatic individuals to produce antibody responses against the surface, microneme and traversal protein of sporozoites following P. vivax natural infection.However, only antibodies to sporozoite surface proteins (PvCSP-VK210 and PvSSP3) could persist in most of our subjects for at least 1 year.Alongside with antibody response to PE antigens, individuals residing in endemic areas have been documented to exhibit elevated antibody responses to asexual blood stage antigens, including PvDBPII, PvMSP8 and PvRPB1a [55][56][57].Therefore, the combination of both PE and asexual stage antigens in multistage vaccine development could be useful for inducing protective immunity against P. vivax infection.
It should be noted that our study has some limitations.A larger sample size with monitoring antibody levels in plasma within days or weeks after infection in the cohort study is required to confirm the kinetics and durability of total and subclass IgG responses to the tested PE antigens.Limitation also includes ascertainment of infection history of participants using records of Vector Borne Disease malaria clinics (Units 11.4.2 and 11.5.3),thereby missing those who had prior mild or asymptomatic infections.Therefore, our study could not demonstrate the relationships between individuals with P. vivax previous infection, as well as magnitude and durability of antibody responses.As repeated infections could boost long-term antibody and MBC responses, examining the dynamics of IgM and IgG antibodies over time and their correlation with memory B cell (MBC) function may provide valuable insights into the humoral immune response against the PE antigen.To gain better understanding of the humoral immunity against PE antigens following natural infection, it is required to establish the baseline of pre-existing IgG responses in individuals with prior P. vivax infections.This data is essential for evaluating the effects of boosting whether current infection could induce IgG levels and investigating correlation with ages, as it was well demonstrated in P. vivaxendemic PNG cohort study [58].
In summary, we demonstrated that natural antibody responses against PE antigens occurred and were persisted after acute vivax malaria.The seropositivity of PE antigens was greatest to full-length PvCSP-VK210 and PvCelTOS, followed by PvM2-MAEBL, PvSSP3, Nand C-termini of PvCSP-VK210, and PvSPECT1 (ranked by frequency of positive responders in cross-sectional study).Naturally-acquired antibody response to PvCSP-VK210 was the most stable seropositivity through one year.PvSSP3 had the longest half-life estimation.The pattern of IgG1 and IgG3 responses was predominant.In-depth study of immunogenicity of these two PE antigens in inducing long-lasting immune responses and their potential association with protection will be useful for the development of effective vivax malaria vaccine.

Fig 2 .
Fig 2. Correlation of antibody responses between specific PE antigens.(A-D) Antibody reactivity correlations between each pair of P. vivax PE antigens (full-length, N-terminal and C-terminal PvCSP-VK210, PvSSP3, PvM2-MAEBL, PvCelTOS and PvSPECT1).Pairwise Spearman's correlations were determined between RI for each antigen at day 0 (acute phase), 90, 270, and 360.Correlation matrices were clustered using the ward.D2 hierarchical agglomerative clustering method.Shown are Spearman's ρ and significance adjusted for multiple testing using the Holm method.* p<0.05; ** p<0.01; *** p<0.001.Spearman ρ>0.60 and adjusted p-value <0.01 are indicative of a strong positive and highly significant correlation.The coloring of the cells represents the sign of correlation: blue gradient for positive correlation and red gradient for negative correlation.The size of background squares represents degree of correlation corresponding to the values shown in each cell.https://doi.org/10.1371/journal.pntd.0011907.g002

Fig 4 .
Fig 4. Half-life estimation of IgG antibodies against PE antigens using an exponential curve fitting model.(A-H) Half-lives of antibodies to PvCSP (full-length, N-terminal and C-terminal subdomains), PvSSP3, PvM2-MAEBL, PvCelTOS, PvSPECT1 and tetanus toxoid.For half-life estimation, antibody responses were normalized as the percentage of maximal antigen-specific total IgG responses across all timepoints and plotted against days after maximal reactivity.Adjusted R 2 (R 2 adj) represents goodness of fit.https://doi.org/10.1371/journal.pntd.0011907.g004